Fig 1.
Summary of disease outcomes in WT recipients of tested BM populations.
Block plot showing the features and diagnoses (rows) for each of the transplanted animals (columns). White blocks indicate the absence and black blocks the presence of each of the features listed. For leukocytosis, thrombocytopenia, anemia, BM hypercellularity and thrombocytosis to be called present, the measured value had to be 2 SD above or below the mean of the WT group. For splenomegaly, a 4-fold cut-off was chosen because in humans a 3–4 fold-size increase represents a “palpable splenomegaly” [25], while BM hypocellularity was defined as <25% of the mean WT value [26]. Disease outcome for each mouse is color-coded above the block plot. Corresponding disease states are indicated below the plot. * Although this mouse presented with thrombocytosis in addition to >10% myelodysplasia, it was not diagnosed as MDS/MPN; it was sacrificed approximately 6 months earlier than the other mice due to a worsening chronic eye infection. This eye inflammation is likely the cause of the moderate leukocytosis (<2SD) and thrombocytosis.
Fig 2.
AML and myelofibrosis in WT mice transplanted with unfractionated Crebbp+/- BM.
(A) Average percentage + SD of donor-derived (CD45.2+) PB leukocytes at the time of sacrifice in recipients transplanted with unfractionated (U) WT or Crebbp+/- BM or with Crebbp+/- LSK, as indicated. Data were pooled from 3–4 independent experiments. See S1 Table for details. (B) Representative histological images of BM touch preparations (i) and bone sections from the tibia (ii-iv) (i) WT control (left) showing abundant erythroid precursors and mature segmented granulocytes. Crebbp+/- bone section (right) showing dramatically decreased erythroid precursors and the presence of >20% blasts. (ii, inset) Crebbp+/- bone section, showing an atypical mitotic figure. (iii) Reticulin stain of bone sections; WT control (left) and Crebbp+/- BM (right) demonstrating in the latter a marked increase in reticulin (black fibers). (iv) Crebbp+/- bone section showing an increase in and thickening of trabeculae. (C) (i) Blood cell counts showing the average ± SD of the indicated parameters in recipients of Crebbp+/- BM that developed AML and WT controls. * p<0.001. (ii-iv) Circulating blasts (ii), nucleated RBCs (iii) and RBC phagocytosis (iv). (D) Spleen from a recipient of Crebbp+/- BM and a WT control. Original magnification: ×60 (Bi, Bii), ×40 (Biii), ×10 (Biv).
Fig 3.
Hematologic characteristics of later-onset myelodysplastic disease in recipients of unfractionated Crebbp+/- BM.
(A) Presented are averages ± SD of WBCs, RBCs, PLTs and spleen weights. Recipients were divided into two groups based on the absence (Group 1) or presence (Group 2) of thrombocytosis, the determining factor for a diagnosis of MDS vs. MDS/MPN-U. * p<0.05 compared to WT control. (B) BM touch preparations (i-ii and iv-v) and PB smears (iii and vi-vii) showing characteristic features of tri-lineage myelodysplasia. (i-iii) Dysplasia of erythroid lineage manifested by karyorrhexis (i, inset and arrow heads) i.e., segmentation of the red cell nucleus and binucleated erythroid precursors (ii, inset and red arrows) as well as anisocytosis (iii). (iv-v) Dysplasia of megakaryocytic lineage indicated by binucleated dwarf megakaryocytes (iv) and multinucleated megakaryocytes (v). (vi-vii) Dysplasia of myeloid cells with pseudo Pelger-Huet anomalies (vi) and hypersegmented granulocytes (i.e. with more than six segments of irregular size) (vii). Original magnification: ×40 (i-ii), ×60 (iii-vii). (C) Survival curve of recipients of unfractionated Crebbp+/- BM transplants.
Fig 4.
Hypercellular BM and tri-lineage myelodysplasia in WT mice transplanted with Crebbp+/- LSKs.
(A) BM section showing a hypercellular BM. (Bi-ii, Civ, Div) PB smears and (Ci-iii, Di-iii) BM touch preparations showing characteristics of tri-lineage myelodysplasia. Dysplasia of the myeloid lineage is indicated by hypersegmented granulocytes (Bi) and pseudo Pelger-Huet anomalies (Bii). Dysplasia of the erythroid lineage was often manifested by binucleation (Ci), an abnormal nuclear contour (Cii) and karyorrhexis (Ciii). Anisocytosis (Civ) and basophilic stippling (Civ,inset) are other features of pathological erythropoiesis found in these mice. Dysplasia of the megakaryocytic lineage is characterized by naked megakaryocytic nuclei (Di), binucleated megakaryocytes Dii), hyperlobulated megakaryocytes (Diii) and giant PLTs (Div,arrow). (E) Atypical localization of immature precursors (i.e., clusters of myeloid precursors present in the intertrabecular area (red dashed line), rather than adjacent to trabeculae or surrounding endothelial cells. Magnification: ×10 (A), ×40 (Civ), ×60 (B, Ci-iii, D-E).
Fig 5.
Increased myelopoiesis of WT origin in WT mice transplanted with Crebbp+/- CMPs or GMPs.
(A) Proportion of Crebbp+/- cell-derived reconstitution (CD45.2+) in the PB of individual WT recipients (CD45.1+) as a function of time. Left panel shows 10 recipients of Crebbp+/- CMPs, right panel shows 14 recipients of Crebbp+/- GMPs. (B) Bar graphs showing the average percentage + SD of (CD45.1+) WT myeloid populations in the BM (upper panel), PB (middle) and spleen (bottom). Black bars represent recipients transplanted with CD45.2+ WT BM, gray bars recipients of CD45.2+ Crebbp+/- CMPs and white bars recipients of CD45.2+ Crebbp+/- GMPs. Measurements were taken at the time of sacrifice, i.e., between 10–19 months (S3 Table). Symbols indicate significant differences with WT; * p<0.05; † p<0.01; ‡ p<0.001. (C) Histological evidence of AML in mouse #5 (S3 Table). (i) BM touch preparation showing the prevalence of blasts (>20%) and the relative lack of maturing trilineage hematopoiesis. (ii) H&E-stained BM section. Arrows and inserts indicate an atypical mitotic figure. (iii) Reticulin staining of BM section. Magnification: ×60 (A-B) and ×40 (C).
Fig 6.
Mature Crebbp+/- myeloid cells produce excess MMP9 and have altered cytokine and gene expression profiles.
(A) Quantitative RT-PCR analysis for genes found differentially expressed in tumor-promoting myeloid cells or in MDS patients. Presented is the ratio of gene expression in Crebbp+/- myeloid populations (n = 7) compared to WT controls (n = 6), purified from the BM of 1 year-old animals. Symbols indicate statistical significance; * p<0.05; † p<0.02; ‡ p<0.01. (B) MMP9 Western Blot analysis (left panel) performed on Gr1loMac1+ cells and corresponding quantification (right panel, average + SD). (C) Heat map of transcripts of the NanoString Immunology Panel differentially expressed (adjusted P-value < 0.05) in Crebbp+/- vs WT Gr1+Mac1+ cells. The table below the heat map represents the gene ontology terms overrepresented versus the platform background and the associated genes. Genes in grey are downregulated; those in red, upregulated.